Serum albumin is the most abundant protein in mammalian sera (35-50 g/l, i.e. 0.53-0.75 mM, in humans) and several strategies to covalently couple a peptide or protein to carrier molecule that will allow in vivo association to serum albumin have been described e.g. in WO91/01743, in WO01/45746 and in Dennis et al (J Biol Chem 277:35035-43, 2002). WO91/01743 describes inter alia the use of albumin binding peptides or proteins derived from streptococcal protein G (SpG) for increasing the half-life of other proteins. The idea is to fuse the bacterially derived, albumin binding peptide/protein to a therapeutically interesting peptide/protein, which has been shown to have a rapid elimination from blood. The generated fusion protein binds to serum albumin in vivo, and benefits from its longer half-life, which increases the net half-life of the fused therapeutically interesting peptide/protein. The half life of serum albumin is directly proportional to the size of the animal, where for example human serum albumin (HSA) has a half life of 19 days and rabbit serum albumin has a half life of about 5 days (McCurdy et al, J Lab Clin Med 143:115, 2004).
Streptococcal protein G (SpG) is a bi-functional receptor present on the surface of certain strains of streptococci and is capable of binding to both IgG and serum albumin (Björck et al, Mol Immunol 24:1113, 1987). The structure is highly repetitive, with several structurally and functionally different domains (Guss et al, EMBO J 5:1567, 1986), more precisely three Ig-binding domains and three serum albumin binding domains (Olsson et al, Eur J Biochem 168:319, 1987). The structure of one of the three serum albumin binding domains in SpG has been determined, showing a three-helix bundle fold (Kraulis et al, FEBS Lett 378:190, 1996, Johansson et al, J. Biol. Chem. 277:8114-20, 2002). A 46 amino acid motif was defined as ABD (albumin binding domain) and has subsequently also been designated G148-GA3 (GA for protein G-related albumin binding and G148 from the strain of Streptococcus from which it is derived).
Artificial variants of GA3-G148 having a very much improved affinity for human serum albumin were developed (Jonsson et al, Prot Eng Des Sel 21:515-27, 2008; WO2009/016043), as well as engineered high affinity variants with reduced immune stimulatory properties (WO2012/004384). The latter were motivated by the fact that a few T- and B-cell epitopes were experimentally identified within GA3-G148 (Goetsch et al, Clin Diagn Lab Immunol 10:125-32, 2003), making this domain as such less suitable for use in pharmaceutical compositions for human administration. Throughout the present text, GA3-G148 as well as the various engineered derivatives thereof presented e.g. in WO2009/016043 and WO2012/004384, are collectively referred to as “ABD”. Thus, in the present disclosure, “ABD” denotes these classes of albumin binding polypeptides, rather than a specified polypeptide with a specific amino acid sequence.
With the increased interest in incorporating an albumin binding domain into therapeutic or diagnostic compositions follows a growing need for inexpensive and efficient purification strategies for isolation of molecules covalently linked to ABD, produced for instance by recombinant expression in prokaryotic or eukaryotic systems, or by direct chemical conjugation to ABD. A general strategy for purification of recombinantly expressed proteins would be to include a commonly used affinity tag such as a polyhistidine tag, a chitin binding protein (CBP), a maltose binding protein (MBP), a glutathione-S-transferase (GST)-tag or a FLAG-tag, and perform a classic affinity separation using commercial resins developed specifically for each tag. However, for certain applications, and in particular for molecules to be used as therapeutics, the end product must be homogeneous. Because the tag would need to be removed, e.g. by enzymatic or chemical cleavage, it is necessary to ensure complete cleavage to obtain a homogeneous product, or to suffer a loss of yield when removing incompletely cleaved product. Both of these issues serve to increase the production cost of the product. Therefore, a more motivated strategy would be to utilize the ABD moiety itself as a purification tag. One example of this would be to couple recombinant albumin to a solid support (see for example Jonsson et al, Prot Eng Des Sel 21:515-27, 2008; Andersen et al, J Biol Chem 286:5234-41, 2011). From a crude solute, compounds comprising an ABD tag are captured by albumin, non-specifically adsorbed contaminants are removed and ABD-tagged compounds are subsequently recovered by disrupting the specific but reversible interaction with albumin. However, albumin is a large natural carrier molecule with several interaction sites for different proteins, fatty acids, sterols, ions etc., and thus, the background binding of both specific and unspecific components may contaminate the recovered sample. Despite the fact that recombinant human albumin has been developed, it is still expensive to include as an affinity ligand in large-scale production of therapeutics, in particular because it is incompatible with standard procedures for cleaning in place, that would have to be applied for the repeated use of the albumin-coupled matrix. Furthermore, harsher elution conditions may be required to recover molecules containing ABD variants with an exceptionally high affinity for albumin. Such conditions may be unfavorable also for the ABD-tagged molecule.
Protein A from Staphylococcus aureus has long been used as an affinity ligand in the industrial production of monoclonal antibodies and Fc-fusion proteins, due to the native affinity of Protein A for the Fc portion of IgG. Protein A in its entirety, as well as the individual Fc-binding domains thereof, have subsequently served as starting points for the rational design of engineered affinity ligands with improved properties. For an introduction, see the papers by Nord K and co-workers in Prot Eng (Nord et al, Prot Eng 8:601-608; 1995) and Nat Biotech (Nord et al, Nat Biotech 15:772-777; 1997).
It is an object of the present disclosure to provide new methods for purification, separation and/or chromatography of protein molecules containing ABD, for example fusion proteins in which ABD is a fusion moiety. It is moreover an object of the disclosure to provide uses for ABD binding agents in biotechnology, for example in protein purification and separation applications.